Mango anthracnose detection method based on whole fruit volatile organic compounds

Abstract

The invention relates to the technical field of postharvest disease monitoring of fruits, in particular to a whole fruit volatile organic compound-based mango anthracnose detection method. According to the detection method provided by the invention, the occurrence condition of anthracnose in the storage and transportation processes of the picked mangoes is judged or monitored by collecting and analyzing volatile organic compound signals related to anthracnose in headspace gas of the whole mangoes, so that the diseased fruits are identified in time, the disease diffusion risk is reduced, and the safety of the mangoes is improved. And the post-harvest management efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of postharvest disease monitoring technology for fruits, and in particular to a method for detecting mango anthracnose based on whole-fruit volatile organic compounds. Background Technology

[0002] mango( Mangifera indica Mangoes (L.) are important tropical and subtropical fruits, prized for their unique flavor and high nutritional value. However, they are susceptible to diseases during post-harvest storage and transportation, leading to decreased marketability and significant economic losses. Anthracnose is one of the most common and damaging post-harvest diseases affecting mangoes. It exhibits latent infection characteristics, often manifesting symptoms as the fruit ripens and softens during post-harvest storage, transportation, and shelf life. Lesions spread rapidly, and in severe cases, can cause mass rot and quality deterioration. In actual storage and transportation, if diseased fruit is not detected and removed promptly, surrounding fruits are more susceptible to contamination and cross-infection, thus amplifying the risk of loss.

[0003] Currently, the detection of mango anthracnose in production mainly relies on regular manual inspections and assessment of visual symptoms. However, in the latent infection or early stages, the fruit surface often lacks obvious lesions or only shows slight, non-specific changes, making timely identification difficult through visual inspection. Furthermore, individually inspecting fruits inside stacks or packaging boxes is not only time-consuming and labor-intensive but also prone to causing secondary mechanical damage. While laboratory-level methods such as pathogen isolation and culture, and molecular biological detection can be used for pathogen identification, they generally suffer from long detection cycles, high operational requirements, high costs, or are not user-friendly for field applications, making it difficult to meet the practical needs of "rapid, continuous, and early" monitoring during storage and transportation. Therefore, developing a detection method that can objectively assess mango anthracnose during storage and transportation and can be used to promptly detect diseased fruit is of great significance.

[0004] Disease infection and the resulting changes in tissue metabolism are often accompanied by the release or compositional changes of volatile organic compounds (VOCs). Compared to methods relying on external phenotypes, volatile components in the headspace of the whole fruit can reflect the internal metabolic state of the fruit and the disease development process to some extent, and have the potential for non-destructive and repeatable collection. Although existing studies have focused on changes in mango volatile components related to ripening, storage treatment, or diseases, specific pathogens (such as *Anthracnose sicca*) remain a concern. Colletotrichum siamense There is still a lack of unified and feasible technical solutions for monitoring whole-fruit volatile biomarkers under infection conditions and for storage and transportation scenarios. In particular, there is a lack of biomarker combinations and discrimination rules that can be used to determine the occurrence of anthracnose caused by *Anthracnose sicca*. Therefore, it is necessary to establish a mango anthracnose detection method based on whole-fruit volatile organic compound signals to achieve disease monitoring and risk warning during post-harvest storage and transportation. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for detecting mango anthracnose based on whole-fruit volatile organic compounds. The method provided by this invention can detect 19 volatile organic compounds associated with mango anthracnose (chemical structural formulas are shown below). Figure 1 The relative content of the signal can be detected to determine whether mango anthracnose has occurred and its course, so as to achieve disease monitoring and risk warning during post-harvest storage and transportation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for detecting mango anthracnose based on whole-fruit volatile organic compounds, comprising: The volatile organic compounds (VOCs) in the headspace of the whole mango fruit were detected. Based on the relative content of different VOCs, the course of anthracnose in the mango fruit was determined. See Tables 1-4 for details. Table 1 Grade I Disease Course

[0007] Table 2 Grade II Disease Course

[0008] Table 3 Grade III Disease Course

[0009] Table 4. Grade IV Disease Course

[0010] The Class I disease stage is defined as: anthrax lesions with an average diameter ≤1.5 cm; The Class II disease course is defined as: 1.5 cm < average diameter of anthrax lesions ≤ 2 cm; The Class III disease stage is defined as: anthrax lesions with an average diameter of 2 cm or less and a mean diameter of 2.5 cm or less. The Class IV disease stage is defined as anthrax lesions with an average diameter > 2.5 cm. The average diameter of the lesion = (D1 + D2) / 2; Where D1 is the maximum diameter of a single lesion, and D2 is the diameter in the direction perpendicular to D1.

[0011] Preferably, the pathogen causing anthrax includes *Anthrax sicca* (Siamese anthrax bacillus). Colletotrichum siamense ).

[0012] Preferably, the anthrax bacteria of Siam includes the anthrax bacteria of Siam with accession number CCTCC NO: M 20241875, HNI2.

[0013] Preferably, the mango to be tested includes the Tainong mango.

[0014] Preferably, the method for detecting volatile organic compounds in the headspace of a whole mango fruit includes: The headspace gas of the whole mango fruit to be tested was collected and detected by gas chromatography-mass spectrometry. Alternatively, a sensor array can be used to collect and analyze signals of the volatile organic compounds in the headspace gas. Alternatively, the volatile organic compounds can be detected by specific adsorption. Alternatively, the volatile organic compounds can be detected by chemical response.

[0015] Preferably, the headspace gas is collected by methods including: solid-phase microextraction adsorption and / or syringe extraction.

[0016] Preferably, when gas chromatography-mass spectrometry is used for detection, the relative content of the volatile organic compounds is characterized by the relative peak area.

[0017] This invention provides the application of the mango anthracnose detection method described above in the postharvest management of mangoes.

[0018] Preferably, the post-harvest management of mangoes includes: monitoring and managing anthracnose during mango storage or transportation.

[0019] Preferably, the management includes: removing diseased mango fruits after anthracnose is detected to reduce the risk of disease spread.

[0020] Beneficial effects: The detection method provided by this invention collects and analyzes the volatile organic compound signals related to anthracnose in the headspace of whole mango fruits, and judges or monitors the occurrence of anthracnose during post-harvest storage and transportation of mangoes, thereby realizing timely identification of diseased fruits, reducing the risk of disease spread, and improving post-harvest management efficiency.

[0021] Biological Preservation Instructions Siamese anthrax bacillus HNI2, classified and named Colletotrichum siamense HNI2, Latin name Colletotrichum siamense It was deposited on August 28, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20241875. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 Chemical structural formulas of 19 volatile biomarkers associated with mango anthrax caused by *Bacillus thuringiensis*; Figures 2-4 The results show the relative peak area levels of 14 volatile biomarkers detected at stage IV of the disease in the headspace of healthy mango fruits and anthracnose-infected fruits. Figures 5-7 ROC curves for distinguishing healthy / disease-infected fruit based on 14 volatile biomarkers detected at stage IV of disease progression; Figures 8-10 This is a schematic diagram showing the changes in the relative peak area of ​​14 volatile biomarkers detected in stage IV of the disease course in the headspace of the whole mango fruit as the disease progresses. Detailed Implementation

[0024] This invention provides a method for detecting mango anthracnose based on whole-fruit volatile organic compounds, comprising: The volatile organic compounds (VOCs) in the headspace of the whole mango fruit were detected. Based on the relative content of different VOCs, the course of anthracnose in the mango fruit was determined. See Tables 1-4 for details. The Class I disease stage is defined as: anthrax lesions with an average diameter ≤1.5 cm; The Class II disease course is defined as: 1.5 cm < average diameter of anthrax lesions ≤ 2 cm; The Class III disease stage is defined as: anthrax lesions with an average diameter of 2 cm or less and a mean diameter of 2.5 cm or less. The Class IV disease stage is defined as anthrax lesions with an average diameter > 2.5 cm. The average diameter of the lesion = (D1 + D2) / 2; Where D1 is the maximum diameter of a single lesion, and D2 is the diameter in the direction perpendicular to D1.

[0025] In one embodiment, the anthrax pathogen includes *Anthrax sicca*. In another embodiment, the *Anthrax sicca* includes *Anthrax sicca* HNI2 with accession number CCTCC NO: M 20241875. In yet another embodiment, the mango to be tested includes *Mango taenia*.

[0026] As one implementation method, a method for detecting volatile organic compounds in the headspace of a whole mango fruit includes: The headspace gas of the whole mango fruit to be tested was collected and detected by gas chromatography-mass spectrometry. Alternatively, a sensor array can be used to collect and analyze signals of the volatile organic compounds in the headspace gas. Alternatively, the volatile organic compounds can be detected by specific adsorption. Alternatively, the volatile organic compounds can be detected by chemical response.

[0027] As one implementation method, the headspace gas is collected by: solid-phase microextraction adsorption and / or syringe extraction.

[0028] As one implementation method, when using gas chromatography-mass spectrometry (GC-MS) for detection, the relative content of volatile organic compounds is characterized by relative peak area. The relative peak area refers to the proportion of the peak area of ​​a particular volatile marker to the total peak area of ​​all volatile organic compounds in the same sample. As another implementation method, the relative peak area is calculated based on the peak areas of each volatile organic compound in the same sample to eliminate the influence of differences in overall volatility intensity between different fruits on the detection results.

[0029] The detection method provided by this invention can be based on 19 volatile organic compounds (chemical structural formulas shown in [reference needed]) associated with mango anthracnose. Figure 1 This invention detects the relative content of signals to assess the onset and progression of anthracnose in mangoes. This includes collecting and analyzing volatile organic compound (VOC) signals related to anthracnose (especially that caused by *Anthracnose sicca*) in the headspace of whole mango fruits. This allows for the assessment and monitoring of anthracnose occurrence during post-harvest storage and transportation, leading to timely identification of diseased fruits, reducing the risk of disease spread, and improving post-harvest management efficiency. The detection method provided by this invention is suitable for monitoring anthracnose at the whole-fruit level during post-harvest storage or transportation of mangoes.

[0030] The detection described in this invention includes all disease detections related to all stages of mango harvesting, including storage and transportation processes. The "detection" and "monitoring" described in this invention can also be referred to as "assessment," "judgment," or "discrimination" in this technical field.

[0031] Based on the above advantages, the present invention provides the application of the mango anthracnose detection method described in the above technical solution in the postharvest management of mangoes.

[0032] As one implementation method, the post-harvest management of mangoes includes: monitoring and managing anthracnose during mango storage or transportation.

[0033] As one implementation method, the management includes: removing diseased mango fruits after anthracnose is detected to reduce the risk of disease spread.

[0034] To further illustrate the present invention, the following detailed description of a method for detecting mango anthracnose based on whole-fruit volatile organic compounds, provided by the present invention, is provided in conjunction with embodiments and accompanying drawings. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Application of volatile markers in distinguishing healthy mango fruits from those infected with anthracnose Test methods Tainong mangoes (Tainong No. 1) of uniform maturity, similar size, and without obvious mechanical damage or pests were selected as experimental materials. After washing with tap water, the fruits were immersed in a 2% sodium hypochlorite solution for 1-2 minutes for disinfection, then immersed in 75% alcohol for 1 minute for disinfection, and finally rinsed with tap water and air-dried. *Anthracnose bacterium tumefaciens* HNI2 (isolated from rotten *Golden Emperor* mango fruit, preservation number CCTCC NO: M 20241875) was cultured on PDA plates until the colony diameter reached approximately 6-8 cm. The leading edge of the colony was used to make holes, and 5 mm diameter bacterial blocks were inoculated onto the equatorial region of the fruit using a sterile punch. Three groups were set up: an untreated blank group (BL), a control group inoculated with sterile water (CK), and an experimental group inoculated with *Anthracnose bacterium tumefaciens* (S), with four replicates in each group. The inoculated mangoes were placed in plastic baskets, wrapped with plastic wrap to maintain moisture, and cultured at 25 ℃ and 95% relative humidity (RH).

[0036] The dynamic changes in mango disease symptoms were observed and recorded. The diameter of lesions perpendicular to each other was measured using the cross-sectional method, and the average value was taken. Lesions with an average diameter greater than 1 cm were defined as diseased. Fruits were classified into five disease stages according to the severity of the disease. Grade 0 diseased fruit: Newly inoculated, no disease symptoms yet; Grade I diseased fruit: average diameter of lesions ≤ 1.5 cm; Grade II diseased fruit: 1.5 cm < average diameter of lesions ≤ 2 cm; Grade III diseased fruit: lesions < 2 cm in diameter and average diameter ≤ 2.5 cm in length; Grade IV diseased fruit: average diameter of lesions > 2.5 cm.

[0037] Whole fruits at different disease stages 0–IV were placed in sealed plastic containers (container volume: mango volume = 4-5:1) at room temperature (20-25℃) for equilibration for 1 h. After adsorption by solid phase microextraction (SPME, DVB / CAR / PDMS fiber) for 30 min, they were detected by gas chromatography-mass spectrometry (GC-MS).

[0038] During the detection process, the processing conditions, extraction time, and detection parameters of each sample were kept consistent to avoid the influence of differences in experimental conditions on the detection results. Qualitative analysis of the detected volatile organic compounds was performed using mass spectrometry library comparison, retention time, and relevant literature data, and the chromatographic peak areas of each volatile component were recorded.

[0039] GC-MC conditions: An Agilent 7890A gas chromatograph was used, equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). High-purity helium (He) was used as the carrier gas at a flow rate of 1 mL / min (constant flow mode). The initial temperature was 40 °C, held for 2 min; then increased to 100 °C at 5 °C / min, held for 0 min; finally increased to 245 °C at 5 °C / min, held until the program ended. The maximum column oven temperature was 325 °C, and the total run time was approximately 51 min. The injection port temperature was 250 °C, using splitless mode. The purge valve opened 5 min after injection, with a purge flow rate of 20 mL / min and a total flow rate of approximately 24 mL / min. Mass spectrometry detection was performed using an Agilent 5975C mass spectrometer detector (MSD), with electron impact (EI, 70 eV) ionization. The ion source temperature was set to 230 ℃, the quadrupole temperature to 150 ℃, and the mass scan range to 35–500 m / z.

[0040] During data processing, the chromatographic peak area of ​​each volatile component is used as the basic signal to normalize the peak areas of different volatile components in the same sample, so as to obtain the relative peak areas of each volatile component in the headspace of the whole fruit, which is used for comparison and analysis between different samples.

[0041] The relative peak area (RPA) is defined as: in, Let be the peak area of ​​the i-th volatile organic compound obtained in chromatographic analysis; It is the sum of the peak areas of all volatile organic compounds in the same sample; n is the number of volatile organic compounds detected in the sample; Test results Using the above method, samples of diseased fruit at stage IV of anthracnose disease were compared with healthy fruit samples for testing. The results are as follows: Figures 2-4 As shown.

[0042] The results showed significant differences in the composition and relative peak area distribution of volatile organic compounds (VOCs) in the headspace of anthracnose-infected and healthy mango fruits. Most fatty acid ester VOCs were significantly elevated in anthracnose-infected fruits, with their relative peak areas generally higher than those in healthy fruits. Conversely, aromatic compounds, monoterpenes, and sesquiterpenes were generally decreased in anthracnose-infected fruits, with their relative peak areas lower than or close to those of healthy fruits. These results indicate that in stage IV of the disease process, anthracnose infection can significantly alter the composition and abundance distribution of VOCs in the headspace of mango fruits, forming a characteristic VOC change pattern distinct from that of healthy fruits. Based on the relative peak area characteristics of various VOC markers, healthy mango fruits can be effectively distinguished from anthracnose-infected fruits.

[0043] Furthermore, using the relative peak area (RPA) of a single volatile biomarker among the 14 volatile biomarkers detected in stage IV of the disease course as a continuous variable, and "healthy fruit / disease-infected fruit" as a binary dependent variable, receiver operating characteristic (ROC) curves for healthy mango fruits and anthracnose-infected fruits were constructed using GraphPad Prism software to evaluate the discriminative ability of each volatile biomarker for identifying diseased fruits. The results are as follows: Figures 5-7 As shown in the figure. ROC analysis results indicate that multiple volatile markers have high discriminative power, with their curves significantly deviating from the random reference line and their areas under the curve (AUC) at high levels. The AUC of some markers is close to or equal to 1, demonstrating excellent ability to distinguish between healthy fruit and anthracnose-infected fruit.

[0044] As anthracnose disease progresses on fruit, the relative contents of various screened volatile biomarkers in the fruit's gaseous environment exhibit clear dynamic changes, as shown in the following trends: Figure 8 – Figure 10 As shown, fatty acid esters such as ethyl butyrate, ethyl hexanoate, and methyl hexanoate maintained low levels in stages 0–II of anthracnose, began to rise significantly in stage III, and reached or remained at high levels in stage IV. In contrast, most sesquiterpene markers showed a decreasing trend as the disease progressed. These results indicate that the screened markers have identifiable dynamic response characteristics at different stages of anthracnose disease, providing a basis for rapid identification of headspace volatiles in whole fruit and for disease course assessment.

[0045] Example 2: Application of volatile biomarkers in monitoring the disease progression of Siamese anthracnose fruit Fruits at different disease stages were selected, and the disease stage classification criteria, fruit gas collection methods, detection methods, and quantitative methods were the same as in Example 1. As the anthracnose disease progressed, the composition of volatile components and their relative peak areas in the headspace of the whole mango fruit showed obvious stage-specific changes. The number of detectable volatile markers gradually increased at different disease stages. Six, six, nine, and fourteen anthracnose-related volatile substances were detected in stages I, II, III, and IV, respectively. The union of the volatile substances detected at each stage yielded a total of 19 volatile compounds, as shown in Tables 1-4.

[0046] Further analysis of 14 volatile biomarkers stably detected in stage IV of the disease revealed that, with the progression of anthracnose, the relative peak area levels of various volatile components in the headspace of the whole mango fruit exhibited distinct stage-specific changes, and the trends of different volatile substances varied across different disease stages. These results indicate that *Anthracnose sirenus* infection can cause systemic changes in the volatile components in the headspace of the whole mango fruit as the disease progresses, thus reflecting the disease's development process and severity.

[0047] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting mango anthracnose based on whole-fruit volatile organic compounds, characterized in that, include: The volatile organic compounds (VOCs) in the headspace of the whole mango fruit were detected. Based on the relative content of different VOCs, the course of anthracnose in the mango fruit was determined, as follows: Grade I disease stage: Grade II disease course: Grade III disease course: Grade IV disease course: The Class I disease stage is defined as: anthrax lesions with an average diameter ≤1.5 cm; The Class II disease course is defined as: 1.5 cm < average diameter of anthrax lesions ≤ 2 cm; The Class III disease stage is defined as: anthrax lesions with an average diameter of 2 cm or less and a mean diameter of 2.5 cm or less. The Class IV disease stage is defined as anthrax lesions with an average diameter > 2.5 cm. The average diameter of the lesion = (D1 + D2) / 2; Where D1 is the maximum diameter of a single lesion, and D2 is the diameter in the direction perpendicular to D1.

2. The method for detecting mango anthracnose according to claim 1, characterized in that, The pathogens causing anthrax include *Anthrax sicca* (Siamese anthrax bacillus). Colletotrichum siamense ).

3. The method for detecting mango anthracnose according to claim 2, characterized in that, The anthrax bacteria mentioned include the anthrax bacteria HNI2 of Siam with accession number CCTCC NO: M 20241875.

4. The method for detecting mango anthracnose according to claim 1, characterized in that, The mangoes to be tested include Tainong mangoes.

5. The method for detecting mango anthracnose according to claim 1, characterized in that, Methods for detecting volatile organic compounds in the headspace of whole mangoes include: The headspace gas of the whole mango fruit to be tested was collected and detected by gas chromatography-mass spectrometry. Alternatively, a sensor array can be used to collect and analyze signals of the volatile organic compounds in the headspace gas. Alternatively, the volatile organic compounds can be detected by specific adsorption. Alternatively, the volatile organic compounds can be detected by chemical response.

6. The method for detecting mango anthracnose according to claim 1 or 5, characterized in that, The headspace gas collection methods include: solid-phase microextraction adsorption and / or syringe extraction.

7. The method for detecting mango anthracnose according to claim 5, characterized in that, When gas chromatography-mass spectrometry is used for detection, the relative content of the volatile organic compounds is characterized by the relative peak area.

8. The application of the mango anthracnose detection method according to any one of claims 1-7 in postharvest management of mangoes.

9. The application according to claim 8, characterized in that, The post-harvest management of mangoes includes: monitoring and managing anthracnose during mango storage or transportation.

10. The application according to claim 9, characterized in that, The management includes: removing diseased mango fruits after anthracnose is detected to reduce the risk of disease spread.

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